Stabilization of materials



United States Patent 2,827,452 STABILIZATION OF MATERIALS HermannSchlenk, Donald M. Sand, and Jerry Ann Tillotson, Austin, Minn.,assiguors to Regents of the University of Minnesota, Minneapolis, Minn.,a corporation of Minnesota No Drawing. Application May 31, 1955 SerialNo. 512,324 14 Claims. (Cl. 260-209) This invention relates to thepreservation and stabilization of organic chemical substances. Moreparticularly this invention relates to the protection of organicchemical materials against deterioration, especially autoxidation, byincluding the materials in a carbohydrate complex.

The discovery that autoxidizable fatty acids are stabilized whenincluded in urea is disclosed in the copending application of HermannSchlenk, one of the joint inventors of this invention, and Ralph T.Holman, Serial No. 189,745, filed October 12, 1950, and now abandoned.With urea, stabilization by inclusion is restricted to fatty acids, ormore generally, to essentially, unbranched molecules since only straightchains preferentially react with urea. Potential physiological uses ofinclusion compounds make desirable the choice of host molecules otherthan urea.

It has now been discovered that autoxidizable substances and others arestabilized by inclusion in carbohydrates such as alphaand beta-dextrins,starches and the like. The mode of autoxidation inhibition is differentfrom that of chain inhibitors such as hydroquinone or similarantioxidants. This is proven by the fact that impure adduct preparationswill autoxidize rapidly although the host molecules, i. e. carbohydratesare present in large amounts.

The principal object of this invention is to provide a method ofpreserving and stabilizing organic chemical materials subject todeterioration or change by including them in carbohydrates by formingcomplexes.

Other objects of the invention will become apparent as the descriptionproceeds.

To the accomplishment of the foregoing and related ends, this inventionthen comprises the features hereinafter fully described and particularlypointed out in the claims, the following description setting forth indetail certain illustrative embodiments of the invention. these beingindicative, however, of but a few of the various ways in which theprinciples of the invention may be employed.

Broadly stated, this invention comprises the method of stabilizing andpreserving organic chemical substances, which are subject to physical orchemical change by reacting these materials with a complex-formingcarbohydrate in amounts and under such conditions as to form complexesor inclusion compounds. The preservation of the qualities of the organiccompounds may be utilized during processing, handling and storing of thematerial before final use. The procedure may be applied to bulkmaterials subject to undesirable chemical and physical change but it isalso applicable on a small scale when the stabilization of someingredient admixed with bulk material is desired. The type of organicchemical compound to which the procedure is applied is not limited byeither structure or chemical characteristics of the compounds. They maybe liquid, solid, or gaseous. The compounds are limited in practicalapplication of the invention to those which are subject toautodeterioration or autochange by autoxidation, radiation,polyme'rization and the like upon standing or in storage. Byautodeterioration and autochange are meant phenomena which arespontaneous or self-occurring such asresult,

from normal storage or exposure to the atmosph re under normalconditions and the like, such as autoiu'dation, volatiiization, etc.

This invention is useful for binding gas or volatile material in a solidand stable form, thus greatly decreasing the volatility. The vaporpressure of the compounds is greatly lowered.

inclusion compounds are a special type of complex. They provide a meansby which one compound can be bound with another suitable chemicalwithout changing the chemical character of either. The molecules are unaltered in their chemical nature. The individual compounds may bereconstituted and readily isolated if the presence of the complexingagent would interfere with the eventual use of the bound material.

A wide variety of complex-forming carbohydrate materials are useful inthe practice of this invention among which may be mentioned:

Alpha, beta, and gamma cyclodextrins: These oligosaccharides, sometimescalled Schardinger dextrins, are more exactly described by thenomenclature as cyclohexaamylose, cycloheptaamylose, andcyclooctaamylose respectively;

Starches, i. e., polysaccharides from various natural sources: Morespecifically, without being exhaustive in this enumeration, wecharacterize the types of starches as they may be used for protectiveinclusion, by mentioning their sources: corn, potato, tapioca, wheat,sago, sweet potato, rice, soybean, waxy maize, lily bulb, banana,wrinkled seed peas or other plant sources; bacterial and protozoalstarches such as dextrans; animal and yeast starches such as glycogen.These starches may be used as they are obtained as genuine substancesafter removal of extraneous materials. Also they may be used partiallymodified, purified or fractionated. As

specific examples, soluble starches are mentioned as provided by theLintner or other processes.

One essential feature for the inclusion reaction bringing aboutprotection is the carbohydrate character of the including material,which provides the necessary chemical affinity for the association withthe moiety included. The only other general requirement is thearchitectural fit between the components which permits the attractingforces to act in a multitude of places distributed over the full lengthof the molecuie included. This architectural fit is inherent in thestructure of thecyclodextrin; polysaccharides, not having cyclicstructures, can adjust themselves to the required shapes due to theflexibility or free rotation of their marco molecules. Bothprerequisites, carbohydrate character as well as flexibility of theirmolecular shape, are found in many other classes of polysaccharides, soamong materials of potential value for inclusion stabilization arementioned: celluloses, glucans, manans, galactamanans, xylans,fructosans, and pectins.

Amylose, for example, essentially is a straight chain polysaccharide.The long carbohydrate chain forms a spiral or helix which holds theguest compound in its empty spiral channel. The helix of the starch hasan adjustable width, i. e. for saturated fatty acids, one turn of thespiral is made up by six glucose units, but the helix can be wider sothat seven or eight glucose units form one complete turn. The helixbecomes wider according to the requirementsof the molecule which isincluded. This mechanism of the inclusion reaction is easiestdemonstrated and explained with amylose. It applies also to branchedchain polysaccharides such as amylopectin or saccharides of intermediatedegree of branching. It is implied that mixtures of difierent typesstarches can be used for protective inclusion also.

Starches are advantageous as host molecules fors aman,

nutrients; vitamins, biologicals and the like in that they arethemselves nutritive. The complexes of this invention provide anexcellent means for the administration and slowrelease of substances.such as vitamins, biologi cals and the like avoidingthenecessity formany repeated dosages.

Alpha-dextrin (cyclohexaamylose) is acyclic oligosaccharide derived frompotato orc orn starch by'enzymatic processes. It consists of six glucoseunits linked together by maltose type linkages. Thus, it is closelyrelated to metabolites of the body and is also apreferential complexingagent for. chemicals of therapeutic or nutritional use. Beta-dextrin(cycloheptaamylose) is V closelyrelated to alpha-dextrm. andpossessesthe same advantages.

The complexes are formed by admixing; the reactants in a solvent-foroneof thelcompounds, either the host or. the guest. Preferably, a mutual' solventis used, 'A.precomplex fOrmatiomtakes placeinsolution;Although. prercomplexing takesplacein most instances byistirring theingredients at room temperature dissolution of the ma-. terials is oftenaccelerated and consequently pro-complex: formationis promoted by theuse of'elevated temperatures up to about 60 to 70."'C. No complex isformed above thesetemperatures. In most instances the complexprecipitates as crystals uponcoolinglto roorn temperatures' or below,although in some cases the precipitant. may not-be crystalline complexinelusion compoundthroughout. For example in some instances where starchis used as the hostmaterial part of the guest molecule. may. be simplyembedded in the host and thereby protected. The precipitant is separatedby .decantation, filtra-.

7 tion, centrifugation or the like and then dried.v

The capacity of the host molecule: to receive aaguest molecule isdependentgupon the: spaces-within the hostmolecules' That.- is, theamount. of' material which can i be bound; and.; protected is determinedvolumetrically Accordingly; no fixed molar ratio betweenthethostandguestmoleculezcan bestatedr When .theguest molecules: are of.materialsihavinga density'of' about one, it may be stated as a generalrule, that starches willbind up. to. about 8%- of theireown; weight of aguest compound and dextrins will receive -,up to.- about 10%. Themechanism is comparablc; to -fillinga box or canor similar container.The container will not-hold more thar'uitsmaximum. capacity. althoughit; may be filled .With anylesseramount.

wheret maximumtutilizafion foflthe .protective. andi staa bilizing;characteristics of ithe; complex :is. to be achieved the- .complex.is;initially formediwith-an excessof the: guest.compoundginordentoinclude as much of the guest-' ElSiPOSSiblB'.Whenthisiis done, in zmanysinstances, part I.

ofithe excess gue'stfmaterialcclings to the. outside of the crystallinecomplex; Onlyuthe material whichis includedjsprotected so;that:foroptimum protection the complex should then.be purified .by' heating-todrive oii the-excess guest material.. This is-preferably done under highvacuum;. r

On the other hand, when something-less-than maximum utilization of: theprotection afiorded by the inclusion compounds; is. not objectionable;thecomplex may be initially formed without an:excess of the guestmolecule in order to.avoidtheI-necessityfor distillation. In eachinstance .the capacity of a-particular complex formin-g carbohydratehostmolecule for anyv particular guest'can Alpha-dextrin. (2' g.) was 5dissolved in? 15 ml. iwateri and mixed withlinoleic,acid;(0.4 g.)diSSOiVed'-in-152;

mlethanol.. The; mixture was-.heatedtto 70. C.. .C omplex formation tookplace upon cooling. After 4 hours the crystals formed were isolated bycentrifugation and by subsequent'suction on'a Buechner funnel.Approximately half of the amount was put in a distilling tube and heatedto l30-150 C. at 0.5 pressure for 16 hours. Someexcesslinoleic acid.condensed'in the coldg inHWarburgvesselsL All manipulations were.carried .out; 7 under nitrogen-sol far. as practicable. to. avoidianyxautoxi dation duringthe preparation. Upon 'immersionof; thevesselslin a,37." C. ;bath they; were flushed: withv pure After 4minutes the system was closed. and

oxygen. the oxygen uptake. was measured. manometrically; and

comparedwith the oxygen uptake of pure linoleicacid Microliter uptakeafter hours Substance Mg.. v

Acid e 2 5 V 10 20'- 30 40 50 It'fis seenithat complex .If'which hadbeen purifiedaby heating; under. high, yacuum afforded virtuallycompleteprotection. Tlie conditions for. the autoxidationare veryrigorousand the value of; microlit'er. after. '50 hours may beconsidered! as equivalent to. a-value afterseveral week's of'exposure toair. In fact, thisLandQothersamples. similarly prepared did, not.exhibitany; signsof, autoxidation after months of.storage inanairfilledstoppered test tube.

The use ofLp ure.v linoleicf acid: as the control tective agents.Exposed'surfaces is one of the factors determining .the rateofautoxidation The large surface of the owdery. adducts thus comparesunfavorably with the .relatively. small expi sed-zsurface. ofthelinoleicacid. a V 'NeVerthelesetherate;.ofi antoxidatiomof the pure ma-=terialin amounts. approximziting vv those in the. adducts were.taken,-as. contrpLrates-in thisand otherexamples-T heating, anabundantamount'of carbon dioxide was re t 65 be determinedempiricallyoby initially. attempting to react 5 fill in an idealway.

leased while the crystalsdissolved; They were stable in 7 aqueoussuspension at room temperature'as microscopic observation. showed; Thecharacteristic shape of the crystals. didnot change over a period. ofhours, and" micro gas bubbles could not be detected in the mother.

liquor. The substance can be isolated by centrifugation and dryingfinairg or bysuctionf After two days of exposure to air, ,itwas analyzedfor carbon dioxide. In.

several preparations the molar ratios carbon dioxide:

alpha-'dextrin. varied-from 1:10 to 7 1:1, corresponding to 0.5% to5%:boundin-th'e dextrin..- Without discuss: V ing. here: the;scientiiio.;hearingofggthis. unexpected comrplex:formation attentionmaybe called to the tact-that the :properties; of tthe.=CO complexvdescribed ,abovefuh j the: requirements :for'its. use.as.baking powder.1

is-a" severe test ofth'eteifectiveness of the complexes as.pro-' Upon"Other alpha-dextrin inclusion compounds have been made with: Vitamin K(Z-methyl naphthoquinone); various fatty acids including butyric,valeric, isobutyric, isovaleric, pelargonic, capric, undecylenic,lauric, myristic, palmitic and stearic; monoand symmetrical dipalmitin;hydrocarbons (Skellysolve F, C, and S, a series of solvents consistingof hexanes, heptanes, octanes and solvent naphtha); and gamma, gamma,gamma-trichloro-beta-oxybutyric acid.

EXAMPLE 3 Beta-dextrin (8 g.) and linoleic acid (1.2 g.) were mixed andheated in 100 m1. of 50 vol. percent aqueous ethanol. The mixture wasstirred for 4 hours at room temperature and then centrifuged. Afterdrying the solids over sulfuric acid, then over phosphorous pentoxide at0.5 mm. pressure, the yield was 7.7 g., 7.3% of its weight beinglinoleic acid. An aliquot of this was put into the center of a Warburgvessel (I, 1.5 g.). A second aliquot was placed in a Warburg vesselcontaining 0.5 ml. water in the sidearm so that the complex was notwetted (II, 1.5 g.). A third aliquot was washed thoroughly withtrimethylpentane and dried at high vacuum. It contained 7.1% linoleicacid (111, 1.5 g.). Another aliquot was treated for 9 hours at 122C./0.5 mm. It contained 6.9% linoleic acid (IV, 1.7 g.). The oxygenuptake of these samples is compared, in Table 2, with that of purelinoleic acid initially used for the preparation (V).

6 (II, 1.1 g.). The'autoxidation of the two samples is compared withthat of pure linoleic acid (III) in Table 3.

Table 3.0xygen uptake of linoleic acid in beta-dextrin at 37 C. under100% oxygen Microllter uptake after hours Substance Mg.

Acid

Again it is seen that the crude complex is not satisfactorily protectedagainst oxygen, whereas the heated product is very resistant.

EXAMPLE 5 Beta-dextrin (1.6 g.) and linolenic acid (0.32 g.) werereacted as already described in 20 ml. of 50 vol. percent aqueousethanol. The mixture was shaken for hour at room temperature. Afterheating for 17 hours at 122 C./0.5 mm. the solid residue weighed 1.45 g.and contained 9.6% linolenic acid. An aliquot of this material wasbrought into a dry Warburg vessel (I, 0.7 g.), another aliquot wasplaced in a vessel containing 0.5 ml. water in the side arm (II, 0.65g.). The oxygen uptake of pure linolenic acid is given for comparisonunder (III) in the following Table 4.

Table 4.Oxygen uptake of linolenic acid in beta-dextrin at 37 C. under100% oxygen Table 2.0xygen uptake of linoleic acid in beta-dextrin at 37C. under .7 00% oxygen Microliter uptake after hours Sub- Mg. stanceAcid As in Table 1, it is seen that the product heated in vacuo ispreserved best (IV). The crude complex showed some protection againstoxygen (I). Surprisingly enough, the effect was greatly increased whenthe crude substance I was tested under humid oxygen (II). This iscontrary to experience, in which humidity is known to be an acceleratorrather than an impediment to autoxidation. I Vhen the crude complex waswashed, the protective quality was somewhat improved (III) but lesssatisfactory than when the product is heated under vacu- EXAMPLE 4 Thecomplex of beta-dextrin and linoleic acid was prepared as before from 5g. and 0.4 g. respectively in 350 ml. water. The product weighed 2.6 g.and contained 8.2% linoleic acid. An aliquot of this material was placedin the Warburg apparatus (1, 1.2 g.). The remainder was subjected to aheat treatment at 122 C./0.5 The linoleic acid content of the residuewas 8.0%

Most striking in this table are the negative values of (II). Obviouslybeta-dextrin is able to bind gas molecules. After about 24 hours, theprocess releasing gas has ended and the pressure curve converts toslowly ascending values indicating an insignificantly slow autoxidation.Extrapolating the ascending branch of the curve, i. e. from between 24and 57 hours towards time 0 permits an assay of the total oxygen uptakeover the whole period. This value does not exceed 200 microliter,whereas the same total value for I is 630. The improved protective 5eiiect in the presence of water is clearly indicated.

EXAMPLE 6 EXAMPLE 7 Beta-dextrin (5 g.) was dissolved in ml. water andcinnamaldehyde (0.9 g.) was added. After shaking for 16 hours at roomtemperature, excess aldehyde could still be detected in the form ofmicroscopic droplets mixed with the crystals. The solids were isolatedin the usual manner and heated at 100140/0.5 mm. for 3 hours. Theresidue contained 12.3% cinnamaldehyde. The substance had the sweet odorand the characteristic p the mixture was .slowly .cooled underagitation.

cording to.the .othenmethodjthe.starchiractionflwas dissolved-in dilute.aqueous potassium hydroxide solution 7 takenup. 1

flavor *of cinnamaldehyde. A sample '(I, 1.0 g2) was takenforftheautoxidation test and itsoxygen uptakeeompared, with v that ;ofpure cinnarnaldehyde. (II).

Table 5.-Oxygert uptake of cinnumuldehyde in betadextrz'n at 37 C. under1 oxygen Mg. Microliter uptake after hours Substance aldehi e I 123 30vi 52, 78 110. 1,070 II 120 1,500 2,750 4,250 5,400

'ga'm ma; gamma-trichloro betafoxybutyric' acid, and vitamin E.

The preparation of the starch complexes was carried out in two ways.Accordingto one methodthe-moiety to be protected was addedtothe'hot.solutiomof-starch indistilledwater (.110 4%concentrationpfistarch)- and and. the. moiety.to.be.protectedIwasadded.Uponneutralization with dilute :acid solutiona fine emulsion forms whichslowly separator out the starch complex. This method can be carried'outat room temperature. "High temperatures are avoided, this beingimportant in particu lar for preparation using vitamin A or itsderivatives.

1 Theanalysisofthe. starch complex was ,made by .dissolvingthematerialin. 2I N potassium.- hydroxide. Vitamin A is thenstillbound tightby[thestarchlin the aqueous liquid phase .an'dflis not available for,eth'er extraction.

Methanol is added vto theclearalkaline,so1ution.to;pre-

cipitate the starch. The vitamin can then be. extracted withether andanalyzed according to' known procedures. EXAMPLE '8 Crystalline "amyloseobtained by butanohprecipitation was suspended in water. The butanol Wasdistilled otf and the volume of aqueous starch solution adjusted to 90ml. of water containing.1.62 g. starch. 10 ml. of. 2 N KOH was added tothemhots'olution andthe mixture cooled under nitrogen. vitamin Aacetatedissolved in 10 mlhethanol are added A under shaking. fTheemulsionwasneutralized with 50% aqueous acetic acid to a pH value of 6.Afterj'hours of shaking the precipitate was centrifuged 0E and driedover KOH in high vacuunn "Thejdry material 'was groundfin a mortar andextracted cold with. carbon tetrachloride This solvent removes excessvitamin which had not been bound but cannot extract vitamin that isincluded-by the solids. Thedry material contained 1.1% vitamimA acetateand proved .to bestable. in .the autoxidation test at 37.in a100f%oxygen, atmosphere. Withr in .4 ;hours, less than l0rnicro1iterofoxygenh ad been 7 emirates Crystalline amylos wasbtainedby. precipitation: with n-amyl alcohol- It was steam distilledito remove the alcohol and adjustedto'a volume of 110 mlzwhich con- Tothe .clar solution .150 mg.

tained 7.2 g. amylose. To the hotsolution 20 m1. "of" 2 N jKOH'wereadded. Fifty-five mlqof this starch solution were diluted toa totalvolume or '110' ml. "Vitamin A=acetate (0.3 g.) 'wasdissolved in 10ml.ethanol and added under shaking. Thefmixturewas neutralized to pH '6iOwith acetic acid. 'After 5 hours shaking it was centrifuged and'the=sol-ids-wcre-"dried -and washed as described above. 'The yield-was 2Z2g. starchcomplex centrifuged and treated as previously described. -yieldof purified complex was 1.82 g. containing 3.2%.

containing 1.65% vitamin A acetate. 115g. ofthis com plex took up 167microlitengfloxygen in 91 hours during autoxidation test.

H r V EXAMPLE-.10 Fifty five ml. 7 of i the starch solution described inErr-- ample'gwere diluted -with'water to 110ml. volume and in thesame-procedure05 g.-vitamin A alcohol-werere: acted With thestarch. Thef'l-iel'd of dried and washed complex was 2.28 g. which contained 0. 89%vitamin A alcohol. 1.5g. of thiscomplex 13.4 mgpvitamin) had anoxygenuptake of '154 microliter oxygen inf-9lhours.

.EXAMPLE 11 Crystal-liner amylosexwas: prepared by. precipitation with,

tertiarysamyl. alcohol, :and treated as described under.

previous: examples. Thesolution-nsed for the reaction with;the vitamin.was '1005ml. 0;2,N.-KOH containing 324% .amy1ose. =VitaminAeacetateimthe amount-of 030 gltwamdissolvediinilt) ml. -,ethanol andshaken to form a fine emulsion. After neutralization withtaeetic acidand shaking for 4 /2 hours, the precipitate was The vitamin A acetate.1.0 g. of this complex (32 mg. vita- *min Altook up 128 microliter ofoxygen in 91 hours.

the solution was then autoclaved at 120 C. for 1 hr.

, EXAMPLE 12 A solution of starch (Merck soluble, according to-Li-ntner);was prepared by adding a suspension of 18 g.

starchin a small amount of water to 30 m1. of boiling water. j 'Boilingwas continued'for several minutes and The clear solution was cooledrapidly inan ice bath to 45 C., at which temperature 10 ml. aq. 2 N KOHwas i added. :This mixture was cooled to room temperature. The totalvolumeof 365 ml. contained=approximately 5% starch and wasalkalinecorresponding .to 0.05 N. A,

solution 013.360 .mg..vitarnin A acetate'in 30 ml. ethanol was. added to,theabove v.solution and the mixture emulsified by shaking. Acetic acidwas added to neutralize-the alkali to a final .pHof .6. .Aftenstoringior1.6 hours, the water was. evaporated and the material eventually driedover alkali. The yellowish solid was ground to about 20 .mesh;particlesize and washed with carbon tetrachloride to yield '16 g. of 1 productcontaining 1.4%

. vitamin 'A in the form of the acetate. "One gram was The tested forautoxidationunder the usual-conditions. oxygen uptakewas,345,.microliter within.64 hours.

EXAMPLE 13 Sixty-sixg. starch (Merck'soL; Lintner)'was dissolved inwater and treated as described before to 'form 'an approximately6%.solution in 0.05 N KOH. .Fromthis stock solution '(1100 1111.) 350m1. containing 21 g.

starch was mixed-with 1 g.. vitamin A acetate dissolved V a in'30 ml.ethanol. After neutralization withacetic acid,

the mixture was shaken 'at room temperature for '16" hours and'thenstored at'Z C...for 4 hours. The pre 7 cipitate was isolated bycentrifilgation and dried. Any

excess :vitamin .A .Wasremovediby. washing with carbon tetrachloride.The total yieldamounted:.to.14:S g; starch containing 3.l vi tamin :A. Asample :containing :31 mg. vitamin, in'the autoxidation test, consumed-31$ 5 microliter oxygen within '15 'hours. -'After' this the sample wasput -uuder oxygen 'under relative humidity at '37 C. U nder suchconditions within' u hours, 260 microliter oxygen were taken up. Thisindicates that the product is most stable under dry conditions, butstill is very well stabilized under humid stress, which would destroythe vitamin itself at a greatly accelerated rate. Final analysis of thissample proved the vitamin A acetate to be essentially unchanged.

The previous examples describing inclusion by means of polysaccharidesmake use of alkaline solutions of starch. This is of advantage in caseswhere the polysaccharide is soluble in neutral medium only withdifliculty. Such procedure, however, is not essential for achieving theprotective elfect. The fact that neutral solutions can 'be used in thepreparations is of importance when materials unstable in alkalinesolution are to be protected. Such are cinnamaldehyde, vitamin Aaldehyde, or others. The following examples compare the two procedures,that is, neutral and alkaline medium for the primary reaction.

EXAMPLE 14 300 ml. of n-butanol was added to the hot aqueous solution of60 g. Takamine starch in 3.1 of water After cooling, the crystallineamylose was collected by centrifugation. The material was suspended thenin water and the residual butanol removed by steam distillation. Beforecooling to room temperature, 160 m1. of 2 N KOH were added so that thetotal was 400 ml. of an 0.8 N alkaline solution. Methyl linoleate (0.4g. in 5 ml. ethanol) was added to the mixture which then was adjusted toa pH value of 6 with dilute 1101. A precipitate is formed immediatelyand the mixture is agitated at room temperature for several hours. Aftercentrifugation the solids were dried and ground to about 20 meshparticle size. Washing with a lipid solvent and subsequent drying toconstant weight yielded 6.2 g. of a product containing 4.05% methyllinoleate. The material did not take up any oxygen during a period of 41hours under the usual testing conditions.

EXAMPLE A butanol fraction was precipitated from 60 g. Lintner starch,as described in the above example for Takamine starch. After removal ofexcess butanol by steam distillation, methyl linoleate was added to thehot neutral solution. A heavy gel was obtained which was treated at roomtemperature with three times its volume of methanol. This proved to beadvantageous for the subsequent drying of the product inasmuch as itremoved the greater part of the water without extracting marked amountsof methyl linoleate bound with the precipitate. After drying in vacuumthe material was washed with a lipid solvent (carbon tetrachloride) toyield 4.6 g. of starch containing 4.0% methyl linoleate. A sample of 2.5g. corresponding to 100 mg. methyl linoleate took up 536 microliteroxygen within 43 hours.

EXAMPLE 16 A solution of 18 g. Lintner starch was prepared in 320 ml.water and autoclaved. One half of this amount (160 ml.) was mixed with450 mg. vitamin A acetate dissolved in 15 ml. ethanol. After standingfor several hours at room temperature and at 2 C. an aliquot of themixture was brought to dryness and the solid residue then washed withcarbon tetrachloride and dried (product A, neutral process).

EXAMPLE 17 The other half (160 ml.) of the original stock solution fromExample 16 was mixed with 5 ml. 2 N KOH. To this, 450 mg. vitamin Aacetate dissolved in 15 ml. ethanol was added and subsequently the pHwas adjusted to pH 6 by means of acetic acid. A precipitate was formedafter storage at 2 C. It was isolated by centrifugation, washed, andtreated as described in the previous example (product B, alkalineprocess).

The products from Examples 16 and 17 contained 2 .4 to 2.5% vitamin Aacetate (2.15 to 2.2% vitamin A). They were subjected to the usualautoxidation test. After a period of 68 hours, 1 g. of product A,neutral process, had taken up 778 microliter oxygen while product B,alkaline process, had taken up 279 microliter oxygen.

Although the alkaline process appears to be advantageous, the productsprepared by the neutral process compare very favorably with respect toprotection against autoxidation as demonstrated with unprotected vitaminA acetate or methyl linoleate.

The following example describes the use of a diiierent typecarbohydrateas a protective component. While Lintner and Takaminestarches are essentially straight chain (amylose) type compounds, waxymaize is a virtually pure branched (amylopectin) type of carbohydrate.

EXAMPLE 18 Waxy maize starch Was purified by extraction with methanoland then dried. A paste of 18 g. of this product was added to 300 ml.boiling water and then autoclaved, as in other preparations. Aftercooling to 45 C., 30 ml. of 2 N KOH was added. Half of this solution wasmixed with 450 mg. vitamin A acetate dissolved in 15 ml. ethanol. Afterneutralizing and storing for several hours, an aliquot was brought todryness. The residue was powdered to a mesh size of approximately 20 andpurified by washing with a liquid solvent. Comparative experimentsshowed that prolonged washing will decrease slightly the content ofvitamin without chang ing the stability of the starch-vitamin compound.A 1 g. sample was tested which contained 2.7% vitamin A acetate (2.4%vitamin A). It proved to be indefinitely stable in the test by notshowing any oxygen uptake at all.

Vitamin A acetate is one of the commonly available forms of vitamin A.The stabilization process is applicable, however, to other derivativesof vitamin A as well.

EXANIPLE 19 Vitamin A cyclohexyl carboxylic acid ester (940 mg. in 30ml. ethanol) was added to 330 ml. of 0.04 N KOH containing 6.0% Lintnerstarch. After neutralization and storage for several hours at 2 C. theprecipitate was collected by centrifuging and dried in vacuum. Purifiedin the usual manner, 12.3 g. starch containing 3.1% vitamin Aester=2.36% vitamin A was obtained. A 1 g. sample took up 324 microliteroxygen within 68 hours under the usual testing conditions.

It is apparent that many modifications and variations of this inventionas hereinbefore set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only and the invention is limited only by the terms of theappended claims.

What we claim is:

1. The method of stabilizing an unstable organic chemical substancewhich comprises forming an inclusion compound by reacting said organicchemical substance with a complex-forming carbohydrate selected from thegroup consisting of starches and dextrins.

2. The method according to claim 1 further characterized in that thematerials are reacted in a solvent for at least one of the materials.

3. The method according to claim 2 further characterized in that thesolvent is a mutual solvent.

4. The method according to claim 1 further characterized in that theunstable included substance is selected from the group consisting ofvitamin A and vitamin A derivatives.

5. A stable vitamin A composition comprising an autoxidizable substanceselected from the group consisting of vitamin A and vitamin Aderivatives included ll. within a complex-forming carbohydrate produced-byilhe method of claim-4.'

F16. Astable vitamin Acompositionaccording,to claim 5 furthercharacterized-in that; saids-autoxidizable substance is. vitamingAacetate. a

7. The method according to c1aim:1 fulfther characterized in that theincluded compound is gaseous.

8. The method according to. claim 7. further "energized in thatthe-included: gaseous compoundiscarbon dioxide.

9. A stable solid composition comprising, a gaseous substance includedwithin a. complex-forming carbohydrate produced by the method of claim'7.

'10. 'A. stablesolid. composition according to claim 9 furthercharacterized inthatthe gaseous: substance is carbon dioxide.

ll.'A stable flavoring composition whichjcomprises an unstable organicchemical .flavoring substance included within a complex-formingcarbohydrate produced by the method of claim 1. a

12. .A stable flavoring .compcsitiohaaccq slinatto 11 .-further,characterized in that;sa1 d; included unstable organic chem c l. fl oing subs a yis innapi ldehyg e eri 'e tha the un ta n;cl d su tance i ach i-d xtrin in a .Qi t -a L Q S fil flli ahwdroxide solution andaddinga yitamin A materiall ct d f o t eg ollpi ns fiin P Yitam A an itmin A derivatives, neutralizing the solution, and separatn ou lin rcsulinacqmpl Abstracts, vol; 45 1951 a es 'vsn d.7512';; id

U s@ @Emmwm" 0% COMMERCE PATENT QFFECE Q? March 18, 1958 Hermann Smlzlenk at all.

It is hereby certified that errer appears in the printed specificationof the above numbered pa'bant requiring correctiop and that the saidLetsers Patent should read as corrected belowa Column '7, line 25 for"butvric" mad butyric column 11, line 8, for "energized" read charact'erized \==*B Signs-:1 and sealed "this 6th day of May 1958,

(SEAL) Atest:

KARI AXLIE ROBERT c. WATSON Conmissioner of Patents Ameetin Officer

1. THE METHOD OF STABILIZING AN UNSTABLE ORGANIC CHEMICAL SUBSTANCEWHICH COMPRISES FORMING AN INCLUSION COMPOUND BY REACTING SAID ORGANICCHEMICAL SUBSTANCE WITH A COMPLEX-FORMING CARBOHYDRATE SELECTED FROM THEGROUP CONSISTING OF STARCHES AND DEXTRINS.